Project Summary Proteomics is dependent on the collection of tandem mass spectra of peptides and proteins to make identifications in database searches. Large-scale proteome analyses have become more comprehensive as instrument scan speeds have increased and ion dissociation methods have improved. Additionally, protein quantitation benefits from the creation of internal standards and data collection methods to acquire sufficient data for accurate quantification. The YRC invented small window data independent acquisition (DIA) for proteomics more than a decade ago and we have innovated and improved these methods. To improve the comprehensiveness and reproducibility of peptide identification and quantitation, DIA methods will be advanced for bottom-up proteomics. Reagents will be developed for general enrichment of peptides for targeted quantitation of peptides. To determine proteoforms of proteins in complexes, strategies employing capillary electrophoresis to separate intact proteins for top down analysis using new DIA data collection strategies coupled to emerging ion dissociation methods for intact proteins will be developed. To advance multiplexed quantitation of peptides in bottom up proteomics and to multiplex ?molecular painting? new reagents will be developed.